Human keratinocyte (HK) motility is a critical event in the process of wound healing. The biological elements that directly influence HK migration are incompletely understood. The microenvironment of the healing skin wound dictates experiments be designed that directly examine elements of the wound environment and evaluate their influence. Our 3 independent, computer-assisted HK migration assays are able to dissect HK motility from HK proliferation. We demonstrated that the type of connective tissue components juxtaposed to the HK has a major influence upon HK migration. In the last cycle, our data support a paradigm in which extracellular matrices (ECMs) can initiate HK migration in the absence of growth factors (GFs), so-called "ECM-initiated" motility or haptotaxis. GFs cannot initiate HK motility. Selected GFs are needed to enhance and optimize ECM-initiated migration. Hypoxia also increases HK migration on ECMs. This is in accordance with human skin wounds treated by semi-permeable occlusive dressings which promote re-epithelialization but make the wound oxygen tension immeasurably low. Therefore, we have identified 3 separable biological elements that profoundly influence HK motility: ECMs, GFs and hypoxia. During the last cycle, we focused on how hypoxia promotes HK motility at the cellular level. We found that hypoxia did notjalter the HK integrin profile, but decreased HK-derived laminins 1 and 5 (2 motility "brakes") and increased the lamellipodia-associated proteins - ezrin, moesin and radixin. It also increased selected matrix metalloproteinases such as MMP-9. We also identified 4 important signaling pathways needed for HK motility--p38 MAP kinase, ERK 1/2, PKC delta and FAK-driven signaling. In this proposal, we wish to determine (i) if hypoxia-enhanced motility is mediated via one or more of the 4 pathways we've identified as necessary for HK migration and if hypoxia-enhanced motility is mediated via ECM-initiated, GF-enhanced motility or both and (ii) which signaling pathways are responsible for cellular, morpholological, structural, mechanisms needed for motility such as focal adhesion turnover, lamillipodia formation, and cell polarization. Lastly, in our Third Aim, we will develop a novel animal model to examine the veracity of our in vitro data. This model will consist of genetically engineered human skin equivalents grafted onto hairless mice.